Process development for the production of an E. coli produced clinical grade recombinant malaria vaccine for Plasmodium vivax

Vaccine 27 (2009) 1448–1453

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Process development for the production of an E. coli produced clinical grade recombinant malaria vaccine for Plasmodium vivax Brian A. Bell a , James F. Wood a , Reeta Bansal a , Hatem Ragab a , John Cargo III b , Michael A. Washington b , Chloe L. Wood b , Lisa A. Ware b , Christian F. Ockenhouse b , Anjali Yadava b,∗ a b

Pilot BioProduction Facility, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States

a r t i c l e

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Article history: Received 24 September 2008 Received in revised form 15 December 2008 Accepted 18 December 2008 Available online 10 January 2009 Keywords: Vivax vaccine cGMP Circumsporozoite protein

a b s t r a c t The global eradication of malaria will require the development of vaccines to prevent infection cause by Plasmodium vivax in addition to Plasmodium falciparum. In an attempt to contribute to this effort we have previously reported the cloning and expression of a vaccine based on the circumsporozoite protein of P. vivax. The synthetic vaccine encodes for a full-length molecule encompassing the N-terminal and C-terminal regions flanking a chimeric repeat region representing VK210 and VK247, the two major alleles of P. vivax CSP. The vaccine, designated vivax malaria protein 001 (VMP001), was purified to >95% homogeneity using a three-column purification scheme and had low endotoxin levels and passed the rabbit pyrogenicity assay. The protein is recognized by monoclonal antibodies directed against the two repeat motifs, as well as polyclonal antibodies. Immunization with VMP001 induced high titer antibodies in mice using Montanide ISA 720. We currently have more than 10,000 doses of purified bulk and 1800 vials of formulated bulk vaccine available for clinical testing and VMP001 is currently undergoing further development as a candidate vaccine to prevent malaria in humans. Published by Elsevier Ltd.

1. Introduction Plasmodium vivax is the major cause of malaria outside of subSaharan Africa and accounts for 132–391 million clinical infections each year which are distributed globally [1]. While deaths are rare compared to the incidence of P. falciparum mortality, there are an increasing number of publications reporting severe disease as a result of P. vivax infection [1,2]. Additionally, P. vivax differs from P. falciparum in the formation of dormant liver stages (hypnozoites) that reactivate several weeks to months after the primary infection to cause symptomatic disease. Thus a vaccine to prevent malaria caused by P. vivax is needed, not only to prevent the morbidity associated with the disease, but also to prevent the potential spread of vivax malaria in areas that are currently non-endemic due to the reactivation of hypnozoites. The circumsporozoite protein (CSP) is present on the sporozoites of all species of Plasmodium [3] and has been the target of vaccine studies in animals and humans. In humans, RTS,S, a vaccine based on the CSP of P. falciparum has elicited significant efficacy in

∗ Corresponding author at: Walter Reed Army Institute of Research, Division of Malaria Vaccine Development, U.S. Military Malaria Vaccine Program, 503 Robert Grant Ave, Room 3W63, Silver Spring, MD 20910, United States. Tel.: +1 301 319 9577; fax: +301 319 7358. E-mail address: [email protected] (A. Yadava). 0264-410X/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.vaccine.2008.12.027

Phase II clinical trials as measured by sterile protection to experimental malaria challenge [4–6]. In pediatric studies in Mozambique adjuvanted RTS,S significantly increased time to infection, reduced clinical and severe disease for 18 months in 1–4 year olds [7,8] and significantly reduced time to infection over a 14 week period in infants [9]. Currently plans are underway to initiate Phase III studies in Africa. Based on its success against P. falciparum, a vaccine based on the CSP of P. vivax might be expected to induce protective efficacy. To date, only one P. vivax vaccine has been tested in human Phase 1 studies [10]. The vaccine based on three P. vivax CSP peptides formulated with Montanide ISA-720 elicited anti-P. vivax sporozoite antibodies in a dose-dependent manner, as well as interferongamma (INF-␥) in most volunteers [11]. The circumsporozoite antigen of P. vivax is dimorphic based on the central repeat region and the two alleles, VK210 and VK247, share no immunological cross-reactivity [12]. In order to produce a vaccine for global use, we have developed a synthetic chimeric vaccine, P. vivax malaria protein 001 (VMP001), based on a Korean isolate, that covers the repeat region of both VK210 and VK247 isolates as well as the flanking N-terminal and C-terminal domains [13]. The vaccine produced in Escherichia coli and formulated with Montanide ISA-720 was immunogenic in mice and elicited antibodies directed to the N-terminal, C-terminal and repeat regions of P. vivax CSP and recognized live sporozoites. Since sera from acutely infected individuals from an endemic area also recognized this pro-

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tein, confirming that not only is VMP001 immunogenic, but that it also retains its antigenic character, we decided to manufacture the vaccine following conditions suitable for testing it in humans. Antigen down selection on its own is not sufficient for the development of a vaccine. It is essential to choose a suitable expression system, a robust purification process, as well as an adjuvant that is compatible with the antigen, and approved for human use, to further the vaccine candidate. Here we report the process development for P. vivax malaria protein 001 (VMP001) produced under current Good Manufacturing Practices (cGMP) at the Pilot Bioproduction Facility (PBF) at the Walter Reed Army Institute of Research. The vaccine produced in E. coli was successfully scaled up and we currently have more than ten thousand doses of clinical grade vaccine available as purified bulk and about 1800 vials of the VMP001 vaccine available as formulated bulk ready for clinical testing. We are currently planning to perform toxicology studies in order to prepare an investigational new drug application for testing in humans.

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sion was confirmed using monoclonal antibodies specific for the repeat region of P. vivax [13]. 2.2. Cell banks After small scale expression studies, a clone was selected for process development and passaged six times on Select APSTM (Alternative Protein Source; Difco, Becton Dickinson, Sparks, MD, USA) agar plates, a plant-derived medium suitable for human use. Glycerol stocks, designated research cell banks, were made and stored at −80 ◦ C. 2.3. Fermentation Initial expression conditions were determined at a small scale and then transitioned to a 10 L BioFlow3000 fermentor (New Brunswick Scientific, Edison, NJ, USA). Expression was carried out in Select APSTM (Alternative Protein Source) broth with 0.8% glycerol, 1% glucose, and 25 ␮g of kanamycin/mL medium. The process was scaled-up to a 300 L fermentor (New Brunswick Scientific, model IF-400, Edison, NJ, USA) under cGMP conditions. After inoculation, the cells were grown to mid-log phase (OD600 of 4.0) and then induced with 0.1 mM IPTG (isopropyl ␤-d-1thiogalactopyranoside) and allowed to grow for another 2 h. The cells were harvested by centrifugation (Sharples tubular bowl centrifuge, model AS-26SP) and the cell paste was aliquoted and stored at −80 ◦ C.

2. Materials and methods 2.4. Purification of bulk VMP001 protein 2.1. Cloning and expression of chimeric CS The rationale and design of the synthetic CS gene construct encoding for VMP001 (Fig. 1a) has been reported previously [13]. Briefly, the amino acid sequence of a Korean isolate of P. vivax was used as a template to construct an E. coli codonoptimized synthetic gene (BlueHeron Biotech, Inc. Bothell, WA, USA). The insert encoding the chimeric CS was subcloned into pQE60-AKI vector [14] in frame with a 3 His6 tag. The resultant plasmid, designated ePvCS1-2-AKI (Fig. 1b), was cloned into E. coli strain XL1-blue cells and plasmid from a positive clone was used to transform E. coli strain BL21(DE3) under kanamycin selection. Expres-

The laboratory-scale purification was carried out using the AKTA Prime (GE Healthcare, Piscataway, NJ, USA) purification system and the flow-rates were converted for the scaled-up production under cGMP. An aliquot of the cell paste was thawed and resuspended in a 20 mM sodium phosphate buffer containing 2 M sodium chloride, pH 6.2 at a buffer:paste ratio of 30:1 (w/v) using a IKA Turrex model T-50 basic homogenizer. After cell disruption (110Y microfluidizer, Microfluidics Corp., Newton, MA, USA) lysed cells were centrifuged and the VMP001 protein was extracted from the soluble fraction using a three-step chromatography procedure. The supernatant was treated with 1% N-lauryl sarcosine (Sigma, St. Louis, MO,

Fig. 1. (a) Schematic representation of VMP001 vaccine construct which encompasses the N-terminal, central repeat and the C-terminal regions of the CSP of P. vivax. Conserved Regions I and II are marked by arrows. (b) Plasmid map showing the gene encoding for VMP001 which was cloned into pQE60-AKI plasmid under kanamycin selection.

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USA) and then loaded onto Ni-NTA Superflow column (QIAGEN, CA, USA) that was previously equilibrated with loading buffer. The column was washed extensively with a total of 45 column volumes of buffers before eluting with 500 mM imidazole in 20 mM sodium phosphate. The eluted protein was loaded onto a Q anion-exchange column, followed by an SP cation-exchange column. Both the ion-exchange columns were sanitized with 2–3 column volumes of 0.2N NaOH, followed by a wash with 10 column volumes of WFI and equilibration with 5 column volumes of 20 mM sodium phosphate, pH 6.2. VMP001 was eluted from the SP column using 117.5 mM sodium phosphate and 150 mM sodium chloride, pH 7.4, sterile filtered, and the protein concentration was determined by using the bicinchoninic acid (BCA) (Pierce, Rockford, IL, USA) method. 2.5. Formulation and vialing To prepare the final formulation, a 250 mL aliquot of purified bulk was diluted using a 5.9% sucrose solution to obtain a final concentration of 100 ␮g/mL VMP001, 17.97 mM sodium phosphate, 22.9 mM sodium chloride and 4.99% sucrose; 600 ␮L aliquots were aseptically transferred to depyrogenated 3 mL Serum Vials (Wheaton, Cardinal Health Dublin, OH, USA). Following lyophilization, vials were sealed with plastic flip off crimp seals and labeled. The Final Container VMP001 (BPR 830-00; Lot #1395) underwent 100% manual inspection for color, appearance and uniformity of the cake; 98% of vials passed inspection. 2.6. SDS-PAGE and Western blot for purity and stability analysis Clarified bacterial lysate, in-process samples and/or purified VMP001 protein, were electrophoresed on 4–20% Bis-Tris sodium dodecyl sulfate (SDS)polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) under reducing or non-reducing conditions using the MES buffer system and the gel was stained with Coomassie blue R250 stain to detect the proteins. For immunological characterization, the samples were transferred electrophoretically onto nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). The membranes were blocked for up to 1 h using nonfat milk in PBS containing 0.1% Tween 20 (PBS-T). After washing with PBS-T the blots were incubated for 1 h at room temperature with the appropriate primary antibody diluted in PBS-T. After washing, alkaline phosphatase-labeled anti-mouse (or anti-rabbit) IgG (Promega, Madison, WI, USA) was added for 1 h. The blots were washed and developed with nitroblue tetrazolium–BCIP (5-bromo-4-chloro3-indolylphosphate) solution (NBT/BCIP; Promega, Madison, WI, USA). For stability analysis, purified bulk samples stored at −80 ◦ C and lyophilized final container stored at 4 ◦ C for various time intervals were tested by Coomassie and Western blotting at specified time intervals. 2.7. Host cell protein analysis The vaccine preparation was tested for the presence of residual host cell protein using a quantitative ELISA to detect E. coli protein following manufacture’s (Cygnus Inc. Redwood City, CA, USA) recommendations. 2.8. Endotoxin content and rabbit pyrogen testing Quantitative determination of endotoxin content was performed using the Limulus amebocyte lysate gel clot assay (Associates of Cape Cod, Falmouth, MA, USA) following the manufacturer’s instructions. Rabbit pyrogen test was performed by Bioreliance (Rockville, MD, USA) following USP guidelines; VMP001 was considered to have met the requirements for absence of pyrogen if no rabbit showed a temperature rise of 0.5 ◦ C or greater above its respective control temperature at any time period. 2.9. Mass spectrometric analysis The identity of the vaccine product was also confirmed by mass spectrometric analysis using Finnigan LTQ-FT mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm × 75 ␮m id Phenomenex Jupiter 10 um C18 reversed phase capillary column at the W.M. Keck Biomedical Mass Spectrometry Laboratory, Biomolecular Research Facility, University of Virginia, Charlottesville, VA (USA). The digest was analyzed using the double play capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in sequential scans. The data were analyzed by database searching using the Sequest search algorithm against NCBI NR. The vaccine was subjected to an additional battery of quality control tests set as release criteria in accordance with FDA guidelines; these are listed in Table 1. 2.10. Vaccine potency Reconstituted final container was diluted in PBS and 3 ␮g protein was formulated with Montanide ISA-720 (Seppic, NJ, USA) at an adjuvant to protein ratio of 70:30. Female C57Bl/6 mice (Jackson Labs, Bar Harbor, ME, USA) were immunized subcutaneously with 100 ␮L of the formulation and bled 28 days post immunization. All studies were performed under IACUC guidelines. Sera were tested for anti-VMP001

antibodies by ELISA. Briefly, wells were coated overnight at 22 ◦ C with 0.04 ␮g VMP001 diluted in PBS, pH 7.4. Plates were washed and blocked with casein for an hour. To determine anti-VMP001 titers, serum samples from individual mice were diluted 1:50 in 0.5% casein buffer and then diluted two-fold down the plate. After a 2 h incubation plates were washed and incubated with peroxidase labeled antimouse IgG (H + L) (Kierkegaard and Perry, Gaithersburg, MD, USA) diluted 1:4000 in 0.5% casein buffer. Reaction was developed using ABTS and optical density was read at A414 after 1 h. Individual mice were considered to have seroconverted if their post-vaccination titers were more than three standard deviations greater than the mean of the control pre-immunization sera.

3. Results We have previously reported the successful cloning, expression and immunogenicity analysis of VMP001, a chimeric recombinant vaccine based on the circumsporozoite protein of P. vivax. For a product to be developed as a potential vaccine, it is essential to have a well characterized construct and a defined and reproducible process. We developed a process that was simple, scalable and capable of producing several thousand doses of clinical grade VMP001 vaccine. Our objective here was to express VMP001 as a soluble protein in E. coli using current Good Manufacturing Practices. We developed and wrote Batch Production Records (BPRs) for each step of the process. Fig. 2 provides an overall flow diagram of the preparation of cGMP batch of purified VMP001 antigen. 3.1. Bacterial growth and fermentation conditions: transition of fermentation from 10 L to 300 L The first step was to establish master cell banks under cGMP conditions. A master seed lot was prepared following BPR 800-00 by inoculating APS broth containing kanamycin and 1% glucose with inoculum from a frozen research cell bank. Master cell banks were prepared from the logarithmic phase cultures (OD600 of 1.0). One vial of the master seed lot (Lot #1333) was used to prepare production cell banks following BPR 801-00 essentially following the same method as above. The BPRs for both the master and production cell banks are archived at the Walter Reed Army Institute of Research Pilot Bioproduction Facility. One hundred vials each of the master (Lot #1333) and production seed lots (Lot #1334) are also stored at the above facility. Both the master and production cell banks were analyzed using criteria set in the BPR to characterize the insert. The identity of the product was confirmed by restriction mapping, and resequencing the plasmid from the production cell bank (data not shown). Bulk cGMP fermentation was performed using BPR 808-00. Three 3 L Fernbach flasks containing 1 L APS medium were inoculated with 100 ␮L of freshly thawed glycerol stocks grown to an OD600 of 1.0. The growth patterns of the 10 L and 300 L cultures were similar as shown in Fig. 3. The 300 L fermentor was inoculated with 3 L of culture (OD600 of 1.0) and the growth monitored till OD600 reached 4. The culture was induced and cells were harvested 2 h post-induction. The 300 L fermentation yielded approximately 6 Kg cell paste (Lot #1345) that was aliquoted and frozen at −80 ◦ C. 3.2. Purification of VMP001 An aliquot of the fermentation paste (Lot #1345) was used to develop a purification scheme which was documented in a batch production record (BPR 828-00). After cell disruption, the paste was centrifuged and the supernatant was treated with 1% detergent to dissociate proteins that were non-specifically associated with VMP001. VMP001 was purified using a 3-column purification process as described earlier. The initial step of Ni-NTA chromatography removed a majority of contaminants as can be seen in Fig. 4, lanes 6–7 when the Ni-NTA eluted protein was run under nonreducing and reducing conditions respectively. Since this protein

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Fig. 2. General method of preparation of clinical grade VMP001.

was intended for use as a human vaccine we included 2 additional steps using ion-exchange chromatography to further purify it. The diluted protein was passed through the Q-ion-exchange resin that bound contaminants, including endotoxin. VMP001 was further purified using an SP (sulfopropyl) cation-exchange resin. A small pH change in the wash buffer enabled further purification (Fig. 4, lane 9) removing low molecular weight contaminant before eluting the protein with no detectable contaminants (Fig. 4, lanes 10 and 11). The eluted protein was filtered through a Millipak 60 0.22 ␮M filtration unit. The final purified bulk antigen (VMP001, Lot 1392) was aliquoted and stored at −80 ◦ C. The protein con-

Fig. 3. Comparison of bacterial growth curves at the Laboratory (10 L) vs. PBF (300 L) scale, represented by open circles and closed squares respectively, shows that they followed identical growth patterns.

tent was determined using the BCA assay and subjected to sterility testing to determine its suitability for formulation under cGMP. An aliquot of the purified bulk protein was diluted to a final concentration of 100 ␮g/mL in 17.97 mM sodium phosphate, 22.9 mM sodium chloride and 4.99% sucrose for the formulation process. Aliquots (600 ␮L) were lyophilized and vialed in a Class 100 cleanroom under BPR 830-00 to yield ∼2000 vials which are stored at 4 ◦ C. 3.3. Characterization of VMP001 The purified bulk (data not shown) and final container (Fig. 5) were also characterized by immunoblotting under reducing and

Fig. 4. In-process samples run on SDS-PAGE and stained with Coomassie blue. The starting sample (lane 1) was subject to a three-step to obtain a pure final product. Lane 2 Ni-NTA flow-through; lanes 3–5 Ni-NTA washes; lanes 6 and 7 non-reduced and reduced VMP001 eluted from the Ni-NTA column; lanes 8 and 9 SP washes; lanes 10 and 11 non-reduced and reduced purified bulk VMP001.

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Fig. 5. Characterization of purified bulk (BPR 828-00, Lot 1392) by immunoblotting. (Panel A) reactivity to mAb 210; (panel B): reactivity to mAb 247; (panel C): reactivity to polyclonal anti-VMP001 serum; (panel D) reactivity to anti-E. coli antibody.

non-reducing conditions using monoclonal antibodies specific to the VK210 (Fig. 5, panel A) and VK247 (Fig. 5, panel B) repeat motifs. Both the monoclonals, as well as the polyclonal anti-VMP001 antibodies ((Fig. 5, panel C), recognize the monomeric form of VMP001 in addition to reduction-sensitive multimers that are seen in the non-reducing lanes. Purified VMP001 did not contain detectable E. coli components as assessed by testing reactivity to polyclonal anti-E. coli antibodies (Fig. 5, Panel D). Additional more stringent testing was performed to look for host (E. coli) contaminants (Table 1). The endotoxin levels in the purified bulk were <0.06 EU/mL VMP001 (Table 1), and final container contained 1.2 EU/mL protein which corresponds to 0.01 EU/␮g VMP001 (data not shown). The sample passed the rabbit pyrogen test following criteria set by USP with no increase in temperature being detected in any rabbit (Table 1). Mass spectrometric analysis of VMP001 showed that the peptides generated correspond to the CSP of P. vivax (data not shown). 3.4. Stability and quality control of VMP001 The purified bulk and final container have been tested for stability at 3-month intervals by SDS-PAGE and immunoblot. A Coomassie stained gel shows that final container is stable at 4 ◦ C up to 1 year (T12 ) and is comparable to the T0 samples (Fig. 6). Purified bulk stored at −80 ◦ C is also stable (data not shown) up to 1 year which is the last time point that has been tested. Several tests were performed on the vaccine in accordance with the FDA guidelines to characterize the product to “to ensure the safety, efficacy, purity and potency of these (vaccine/biological) Table 1 Characterization of purified bulk VMP001 (BPR 828-00, Lot 1395). Test

Result

Sterility Protein content Endotoxin (LAL) Rabbit pyrogen Host cell protein DNA content IPTG Imidazole Heavy metal Kanamycin

No-growth 654 ␮g/mL <0.06 EU/mL Non-pyrogenic <2 ng/50 ␮g VMP001 <40 fg/␮L <0.05 ␮g/mL 234 nmol/mL <0.002% None

Fig. 6. Vaccine stability. Coomassie blue stained reduced and non-reduced VMP001. The final container (Lot 1395) stored at 4 ◦ C for 1 year (T12 ) did not show any appreciable difference from the T0 sample.

Fig. 7. Vaccine potency. All 10 C57Bl/6 mice seroconverted after a single immunization with VMP001 in Montanide ISA 720 with a mean titer (OD414 = 1.0) of 24,000. Dots represent titers of individual mice.

products” (http://www.fda.gov/Cber/vaccines.htm). These included measurement of residual DNA, antibiotic, IPTG and other in-process materials. The results of additional quality control tests conducted to meet the release criteria are shown in Table 1. 3.5. Vaccine potency VMP001 in Montanide ISA 720, an adjuvant that has been used in humans, induced seroconversion in mice (Fig. 7) with a mean titer, calculated as the dilution of serum giving an OD414 of 1.0, of 24,000 after a single immunization (minimum titer 7256 and maximum titer 50,072; geometric mean 19,885), and increased to >150,000 (range 41,979–488,367) following a second dose (data not shown). 4. Discussion Selection of an antigen is an important aspect for the development of a vaccine. For a vaccine that aims to prevent or delay infection caused by malaria parasites the rational antigens to begin with are those present on the surface of sporozoites and liver stages that can be used singly, or in combination with other antigens. The dominant antigen present on the surface of sporozoites has proven

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to be a strong vaccine candidate for malaria caused by P. falciparum and is ready to undergo Phase III clinical trials. The other critical factor for the development of any vaccine is the ability to produce the vaccine with a process that is amenable to scale-up, is reproducible and produces a reasonable number of doses of the vaccine. According to BC Buckland “Process development is the technological foundation that underlies the manufacture of new vaccines and is central to successful commercialization” [15]. Based on the success of the only subunit vaccine for malaria, we designed a vaccine based on the CSP of P. vivax. We have previously reported its immunogenicity and antigenicity [13]. In this manuscript, we describe the development by fermentation, purification and immunogenicity of a candidate recombinant P. vivax CSP vaccine (VMP001) that, after further safety testing, may be suitable for human vaccine trials. Prior to scale-up, it is essential to ensure the reproducibility of the process to ensure its robustness. We ran several fermentations and purifications to ensure that the process was easily translated from the laboratory setup to the large scale cGMP production. Using this defined process VMP001 was purified free of host E. coli contamination, and contained endotoxin (0.5 EU/50 ␮g dose/70 kg human) that were well below FDA standards (350 EU/dose/70 kg human). Using stringent purification conditions we ensured that there was little to no contamination by all in-process chemicals as is required for a vaccine meant for human use per FDA and ICH guidelines. The purified product has remained stable for at least 1 year when assessed by Coomassie staining and immunoreactivity. Unlike drugs which have are usually small molecules with a defined structure, vaccines are larger molecules which are much more complex and, therefore, more difficult to characterize. Thus, the most important aspect of the characterization of a vaccine is defining the product by virtue of its reactivity to antibodies (preferably monoclonal) and its potency in animals. VMP001 has a conformational structure that allowed recognition by two mAbs to each of the P. vivax CSP alleles, as well as eliciting anti-VMP001 antibodies in mice that agglutinated P. vivax sporozoites [13], suggesting that these antibodies may confer protection. Since VMP001 is also recognized by sera from individuals naturally exposed to P. vivax infection, we conclude that VMP001 is a strong candidate for a malaria vaccine designed to elicit protective anti-P. vivax sporozoite antibodies in humans when adjuvanted with a human use acceptable adjuvant. The vaccine conforms to criteria that have been recommended by the FDA for a product to undergo human testing and we are currently in the process of writing an Investigational Drug Application to conduct a Phase 1 safety study in humans. Acknowledgements Authors would like to thank Joan Lang, Rick Millward, Stacia Moreno, David Bradley and Kenneth E Eckels from the PBF, WRAIR;

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Lee Shirkey, Daniel Kohli and Marcus Ville Titto from the Penn State University Co-op program for their invaluable support during the course of this study; and Dr. Michael Hollingdale for his help during the final stages of manuscript preparation. This study was supported by the Medical Infectious Diseases Research Program of the U.S. Army Medical Research and Materiel Command, Fort Detrick, Maryland. The views and opinions expressed in this study are those of the authors and should not be construed as the official opinion of the U.S. Departments of Defense, or the U.S. Army. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals (NRC Publication, 1996 ed.). References [1] Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria: neglected and not benign. Am J Trop Med Hyg 2007;77:79–87. [2] Baird K. Neglect of Plasmodium vivax malaria. Trends Parasitol 2007;23:533–9. [3] Nussenzweig V, Nussenzweig RS. Circumsporozoite proteins of malaria parasites. Cell 1985;42(2):401–3. [4] Stoute JA, Slaoui M, Heppner DG, Momin P, Kester KE, Desmons P, et al. A preliminary evaluation of a recombinant circumsporozoite protein vaccine against Plasmodium falciparum malaria. RTS,S Malaria Vaccine Evaluation Group. N Engl J Med 1997;336(2):86–91. [5] Kester KE, Cummings JF, Ockenhouse CF, Nielsen R, Hall BT, Gordon DM, et al. Phase 2a trial of 0 1 and 3 month and 0 7 and 28 day immunization schedules of malaria vaccine RTS S/AS02 in malaria-naïve adults at the Walter Reed Army Institute of Research. Vaccine 2008;26(18):2191–202. [6] Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Hall T, et al. Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. J Infect Dis 2001;183(4):640–7. [7] Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J, et al. Efficacy of the RTS S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet 2004;364:1411–20. [8] Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Aide P, et al. Duration of protection with RTS S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial. Lancet 2005;366:2012–8. [9] Aponte JJ, Aide P, Renom M, Mandomando I, Bassat Q, Sacarlal J, et al. Safety of the RTS S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. Lancet 2007;370:1543–51. [10] Herrera S, Corradin G, Arévalo-Herrera M. An update on the search for a Plasmodium vivax vaccine. Trends Parasitol 2007;23(3):122–8. [11] Herrera S, Bonelo A, Perlaza BL, Fernández OL, Victoria L, Lenis AM, et al. Safety and elicitation of humoral and cellular responses in Colombian malaria-naive volunteers by a Plasmodium vivax circumsporozoite protein-derived synthetic vaccine. Am J Trop Med Hyg 2005;73(5 Suppl.):3–9. [12] Rosenberg R, Wirtz RA, Lanar DE, Sattabongkot J, Hall T, Waters AP, et al. Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax. Science 1989;245(4921):973–6. [13] Yadava A, Sattabongkot J, Washington MA, Ware LA, Majam V, Zheng H, et al. A novel chimeric Plasmodium vivax circumsporozoite protein induces biologically functional antibodies that recognize both VK210 and VK247 sporozoites. Infect Immun 2007;75(3):1177–85. [14] Yadava A, Ockenhouse CF. Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun 2003;71(9):4961–9. [15] Buckland BC. The process development challenge for a new vaccine. Nat Med 2005;11(4 Suppl):S16–9.